Useful protein or polypeptide may be produced by a synthesis or isolated from natural sources. However, these methods have disadvantages in that it is uneconomical in terms of cost and time and an output thereof is limited. Therefore, it is preferable that target protein and target polypeptide are produced through a culture of transformant prepared by a recombination in order for the target protein or target polypeptide to be over-expressed. However, polypeptide (especially, short polypeptide) produced under cell environment may be sensitive to a degradation due to a function of protease present in a cell, or since not all antibodies to all the target proteins are present, it may be difficult to purify the target proteins without the corresponding antibodies.
In order to overcome such a problem, a protein tagging or epitope tagging has been used. The protein tagging or epitope tagging is a recombinant DNA method, which includes preparing a recombinant nucleic acid molecule prepared by ligating a coding sequence of epitope tag to a coding sequence of target protein and expressing the recombinant nucleic acid molecule in a proper host cell, and is used for detecting, quantifying, or purifying the target protein using an antibody to the epitope tag, or determining a location of the target protein in a cell, confirming functionality, and the like. Many epitope tags and antibodies that are ligands of the epitope tags are commercially available, and in the case of selecting suitable epitope tags and antibodies thereof, target proteins can be detected or purified by using Western blot analysis, immunoprecipitation, immunofluorescence, immunocytochemistry, immunoaffinity purification, and the like. Therefore, it is unnecessary to produce the antibody to the target protein.
Recently, as an epitope tag used for a protein tagging or epitope tagging, it is possible to use many unique tags such as short peptide tag having about 6 amino acid residues (for example, 6×His tag) or large protein having about 40 kDa (for example, MBP) [see Stevens, R. C., (2000) Structure Fold Des 8:R177-85]. Generally, peptide tag composed of 3 to 30 amino acids is used. His-tagged protein is specifically trapped on a Ni-NTA (nickel-nitrilotriacetic acid) resin, and may be eluted by using EDTA or imidazole. In addition, maltose-binding protein (MBP, 396 amino acids, 40 kDa), staphylococcus protein A, calmodulin-binding peptide (CBP, 26 amino acids, 2.96 kDa), GFP (238 amino acids, 27 kDa), and glutathione-S-transferase (GST, 211 amino acids, 26 kDa) may be used for detecting or purifying procaryotic proteins and eukaryotic proteins. Furthermore, epitope tag that is generally most often used may include a c-myc tag, a HA tag, a FLAG tag, and the like. The c-myc tag is an epitope tag having 10 amino acids length derived from human c-myc protein [Evans et al., (1985) Mol. Cell. Biol., 12: 3610-3616], and the HA tag is an epitope tag having 9 amino acids length derived from influenza hamagglutinin HA-1 protein [Field et al., (1988) Mol. Cell. Biol., 8: 2159-2165]. The FLAG tag is an epitope tag having 8 amino acids length derived from a bacteriophage T7 [Hopp et al., (1988) Bio/Technology, 6: 1204-1210].
Meanwhile, it is preferable that an epitope tag used for tagging an epitope minimally influence on a three-dimensional structure and biological activity of target protein when fusing with the target protein. However, a long epitope tag (for example, a GST tag or a MBP tag) has generally a problem in that it allows functions of the target protein to be changed. On the other hand, epitope tags having a relatively short length, for example, a FLAG tag, a c-myc tag, and the like scarcely influence on properties of target protein fused therewith, can be very specifically bound to antibodies therefor, and do not have to be removed from the fusion proteins on some occasions. Thus, they are mostly used now. However, there are problems in that since the amino acid sequences of c-myc tag and FLAG tag are included in many proteins among the proteins in cells of organisms that are known up to now, non-specific reactions of antibodies recognizing the tags are induced and interrupt an isolation and confirmation of specific target proteins, resulting in decreasing reliability of the experiments [Ksenija Gasic et al., (2005) Plant molecular biology reporter 23:9-16]. In order to overcome the non-specific reaction problem, an affinity purification system has been developed, in which for such an affinity purification system, two different tags are sequentially fused to N-terminal of protein, and then used [Rigaut, G., et al. (1999) Nat Biotechnol 17:1030-2].
Accordingly, it is needed to develop novel peptide tags capable of removing non-specific reaction that is a problem in the conventional epitope tagging system for detecting and purifying a fusion protein expressed in a recombinant host cell using an epitope tag and antibody thereto, and having short amino acid sequence at the same time, and an antibody capable of being used in pairs with the novel peptide tags.